Our long term goal is to understand the regulation of cyclic AMP (cAMP) formation by isoforms of adenylyl cyclase (AC) in the heart, vasculature, and brain. Cyclic AMP plays a key role in sympathetic control of vasodilation, heart rate, and force of contraction. Aberrations in cAMP contribute mightily to hypertension, cardiomyopathies leading to heart failure, and numerous brain abnormalities. The isoforms of AC expressed in vascular smooth muscle and cardiac tissue (AC V and VI) are stimulated by receptors such as the B1 or B2 adrenergic receptors coupled to GaS, and inhibited by receptors coupled to Gai, or by other signaling molecules such as protein kinase A (PKA), and Regulators of G protein Signaling (RGS2). In addition, the subcellular localization of specific AC isoforms appears to be of great importance. In support of this we have identified a complex of AC V/VI and PKA scaffolding proteins known as AKAPs (A-kinase anchoring proteins). Also, our studies suggest that GaS and Gai regulate AC catalytic activity by influencing the conformation of the interface between the cytoplasmic domains of AC. In this proposal, we will examine the mechanism of 3 inhibitory regulators of AC; Gai, RGS2, and PKA/AKAP. We hypothesize that these negative regulators of AC bind to the C1 domain and stabilize an open conformation of AC in direct opposition to AC activators and that AKAP binding and anchoring of AC leads to additional sensitivity to inhibition by PKA and other cellular regulators. Our specific aims are to: (1) Determine the structural determinants for specificity among AC isoforms for Gai mediated inhibition and characterize the AC conformation stabilized by Gai, (2) Identify the RGS2 binding site(s) on the C1 domain of AC and the conformational states of AC capable of binding RGS2, and (3) Elucidate the role of AKAP binding and localization of AC on cAMP production and the potential for interactions between ACs and AKAP family members expressed in the vasculature and heart.